Fastgene plasmid mini kit

250 µL of super optimal broth with catabolite repression (SOC) medium (20 g L⁻¹ Bacto Tryptone, 5 g L⁻¹ Bacto Yeast Extract, 10 mM NaCl, 2.5 mM KCl, and 0.2 M glucose) was added to the mixture, and cells were cultured for recovery at 37 °C for 1 h. The cultured cells were transferred to Luria Bertani (LB) agar medium (10 g L⁻¹ Bacto Tryptone, 5 g L⁻¹ Bacto Yeast Extract, 10 g L⁻¹ NaCl, and 15 g L⁻¹ agar) containing appropriate antibiotics and incubated at 37 °C on a shaker at 220 rpm overnight. Colonies were then transferred to fresh LB agar medium containing appropriate antibiotics, and a master plate was produced. Subsequently, the transformed E. coli were cultured in liquid LB medium (10 g L⁻¹ Bacto Tryptone, 5 g L⁻¹ Bacto Yeast Extract, and 10 g L⁻¹ NaCl) containing appropriate antibiotics, and plasmid DNA was isolated using the FastGene Plasmid Mini Kit (NIPPON GENE, Tokyo, Japan).

The RT-PCR products in Fig. 1b were purified using a QIAquick Gel Extraction Kit (QIAGEN), and the DNA fragments were cloned using a Zero Blunt TOPO PCR Cloning Kit (Invitrogen). The vectors were transformed into Escherichia coli DH5α competent cells (TOYOBO Co. Ltd., Osaka, Japan). Plasmid DNAs were randomly selected from colonies and then extracted using a FastGene Plasmid Mini Kit (Nippon Gene). The inserts were amplified by PCR using M13 forward and reverse primers (Invitrogen) and sequenced using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and a 3130xl Genetic Analyzer (Applied Biosystems).